Biochemistry Ldh Assay Essay example

3623 Words Apr 6th, 2016 15 Pages
BIOL 3380 Name:_____________________________________

Circle Session: T-PM W-AM W-PM R-AM R-PM F-AM F-PM

Experiment 9 – Pre-lab Homework
Enzyme Kinetics of LDH
This pre-lab homework assignment is due at the beginning of your lab session.

You are provided with the following portion of a protocol: • Determine concentration of enzyme stock solution, if unknown, by taking an A280 nm reading of a 1:100 dilution (in water). Use a total volume of 1 ml in the cuvette.

• Dilute some of the enzyme stock with buffer A to make a 4 mg/ml solution.

• Serially dilute the 4 mg/ml solution with buffer A to make working solutions of 400 µg/ml and 40 µg/ml.

• Prepare 30 µl of each working solution for
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a. Calculate what is needed to make the 4 mg/ml solution

b. Calculate what is needed to make the 400 ug/ml working solution

c. Calculate what is needed to make the 40 ug/ml working solution

4. (1 pt) You compare the data from your assay to data obtained on a previous day by the lab’s star graduate student. You recorded a reading of 0.020 ΔA340/5 secs. The grad student recorded a reading of 0.160 ΔA340/min. Whose sample had more LDH activity? (Show your work for credit.)

5. (1pt) Looking at the “Reaction Rate as a Function of Enzyme Concentration” section in the lab protocol, which assay would you predict to have the highest LDH activity and why?

6. (1pt) Looking at the “Reaction Rate as a Function of Substrate Concentration” section in the lab protocol, which assay would you predict to have the highest LDH activity and why?

Experiment 9

Enzyme Kinetics of LDH

You are in a position to characterize the LDH enzyme as a catalyst. This is done by measuring the kinetics of enzyme reactions. (kinetics means the rate of change, or reaction rate). First, a review of enzyme basics:

I. Definitions

Enzymes increase the rate at which a reaction occurs. Enzymes are not consumed by the reaction. They do not shift the equilibrium of the reaction. Substrates are substances acted upon by an enzyme.

II. Properties of Enzymes A. The majority of known enzymes are proteins (some nucleic acids with

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